A novel approach appropriate for rapid separation and immobilization of a single cell by concomitantly utilizing laser manipulation and locally thermosensitive hydrogelation is proposed in this paper. We employed a single laser beam as optical tweezers for separating a target cell and locating it adjacent to a fabricated, transparent micro heater. Simultaneously, the target cell is immobilized or partially entrapped by heating the thermosensitive hydrogel with the micro heater. The state of the thermosensitive hydrogel can be switched from sol to gel and gel to sol by controlling the temperature through heating and cooling by the micro heater. After other unwanted cells are removed by the high-speed cleaning flow in the microchannel, the entrapped cell is successfully isolated. It is possible to collect the immobilized target cell for analysis or culture by switching off the micro heater and releasing the cell from the entrapment. We demonstrated that the proposed approach is feasible for rapid manipulation, immobilization, cleaning, isolation and extraction of a single cell. The experimental results are shown here.

Background:The mechanism of H5N1 pathogenesis in humans remains unclear. Results: SAEC-T clones were poorly susceptible to previously circulating avian influenza viruses but were completely susceptible to H5N1. Conclusion: Infectivity depends on a delicate balance between acid stability of viral hemagglutinin and endosomal pH in infected cells. Significance: These findings could explain why H5N1 is directly transmitted to humans from birds, resulting in serious illness.

A major problem when analyzing bionanoparticles such as influenza viruses (approximately 100 nm in size) is the low sample concentrations. We developed a method for manipulating a single virus that employs optical tweezers in conjunction with dielectrophoretic (DEP) concentration of viruses on a microfluidic chip. A polydimethylsiloxane microfluidic chip can be used to stably manipulate a virus. The chip has separate sample and analysis chambers to enable quantitative analysis of the virus functions before and after it has infected a target cell. The DEP force in the sample chamber concentrates the virus and prevents it from adhering to the glass substrate. The concentrated virus is transported to the sample selection section where it is trapped by optical tweezers. The trapped virus is transported to the analysis chamber and it is brought into contact with the target cell to infect it. This paper describes the DEP virus concentration for single virus infection of a specific cell. We concentrated the influenza virus using the DEP force, transported a single virus, and made it contact a specific H292 cell.

Mammalian cells must produce heat to maintain body temperature and support other biological activities. Methods to measure a cell's thermogenic ability by inserting a thermometer into the cell or measuring the rate of oxygen consumption in a closed vessel can disturb its natural state. Here, we developed a noninvasive system for measuring a cell's heat production with a bimaterial microcantilever. This method is suitable for investigating the heat-generating properties of cells in their native state, because changes in cell temperature can be measured from the bending of the microcantilever, without damaging the cell and restricting its supply of dissolved oxygen. Thus, we were able to measure increases in cell temperature of <1 K in a small number of murine brown adipocytes (n = 4-7 cells) stimulated with norepinephrine, and observed a slow increase in temperature over several hours. This long-term heat production suggests that, in addition to converting fatty acids into heat energy, brown adipocytes may also adjust protein expression to raise their own temperature, to generate more heat. We expect this bimaterial microcantilever system to prove useful for determining a cell's state by measuring thermal characteristics.

We developed an active virus filter (AVF) that enables virus enrichment for single virus infection, by using insulator-based dielectrophoresis (iDEP). A 3D-constricted flow channel design enabled the production of an iDEP force in the microfluidic chip. iDEP using a chip with multiple active virus filters (AVFs) was more accurate and faster than using a chip with a single AVF, and improved the efficiency of virus trapping. We utilized maskless photolithography to achieve the precise 3D gray-scale exposure required for fabrication of constricted flow channel. Influenza virus (A PR/8) was enriched by a negative DEP force when sinusoidal wave was applied to the electrodes within an amplitude range of 20 Vp-p and a frequency of 10 MHz. AVF-mediated virus enrichment can be repeated simply by turning the current ON or OFF. Furthermore, the negative AVF can inhibit virus adhesion onto the glass substrate. We then trapped and transported one of the enriched viruses by using optical tweezers. This microfluidic chip facilitated the effective transport of a single virus from AVFs towards the cell-containing chamber without crossing an electrode. We successfully transported the virus to the cell chamber (v = 10 µm/s) and brought it infected with a selected single H292 cell.

f Photoautotrophic bacteria have developed mechanisms to maintain K ؉ homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K ؉ uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K ؉ (Kdp). The contributions of each of these K ؉ transport systems to cellular K ؉ homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K ؉ uptake, albeit with lower rates than were conferred by Ktr. An onchip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K ؉ uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K ؉ depletion. Correspondingly, Kdp-mediated K ؉ uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K ؉ required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K Living cells have developed specific responses to hyperosmotic shock. Upon exposure to this stress, cells initially lose water and their volume shrinks. In all living cells, K ϩ is the major intracellular cation used for the maintenance of turgor pressure, cytosolic osmolarity, protein structuring, and membrane potential (1-3). In contrast to animals, Na ϩ /K ϩ ATP pumps are generally missing in bacteria and plants. Hence, these cells possess K ϩ uptake transporters to supply K ϩ to the cells. Particularly after hyperosmotic stress, cells quickly take up K ϩ from the medium to increase the intracellular osmolarity, which prevents water efflux from the cell. Data from genetic and biochemical experiments indicate that the activity and the expression of these transporters respond to hyperosmotic stress. In the later phase of acclimation to hyperosmotic stress, cells also induce the synthesis of osmoprotective molecules, such as glutamate, trehalose, proline, and glucosylglycerol (4, 5). Despite an increasing amount of data on cellular osmoregulation involving ion flux across the membrane, direct evidence for the involvement of specific transporters in the cellular response to osmotic up-shock is lacking for photoautotrophic organisms.The cyanobacterium Synechocystis sp. strain PCC 6803 (hereinafter referred to as Synechocystis) is a frequently used unicellular photosynthetic prokaryote that can survive under a wide range of environmental conditions (6). Unlike Escherichia coli, Synechocystis possesses an internal thylakoid membrane system, which...

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Influenza virus infection can result in changes in the cellular ion levels at 2–3 h post-infection. More H+ is produced by glycolysis, and the viral M2 proton channel also plays a role in the capture and release of H+ during both viral entry and egress. Then the cells might regulate the intracellular pH by increasing the export of H+ from the intracellular compartment. Increased H+ export could lead indirectly to increased extracellular acidity. To detect changes in extracellular pH of both virus-infected and uninfected cells, pH sensors were synthesized using polystyrene beads (ϕ1 μm) containing Rhodamine B and Fluorescein isothiocyanate (FITC). The fluorescence intensity of FITC can respond to both pH and temperature. So Rhodamine B was also introduced in the sensor for temperature compensation. Then the pH can be measured after temperature compensation. The sensor was adhered to cell membrane for extracellular pH measurement. The results showed that the multiplication of influenza virus in host cell decreased extracellular pH of the host cell by 0.5–0.6 in 4 h after the virus bound to the cell membrane, compared to that in uninfected cells. Immunostaining revealed the presence of viral PB1 protein in the nucleus of virus-bound cells that exhibited extracellular pH changes, but no PB1 protein are detected in virus-unbound cells where the extracellular pH remained constant.